PROJECT SUMMARY This proposal aims to develop a novel caveolae targeting antibody for rapid clinical translation. Its long-term objective is to develop the caveolae pumping system, an active transendothelial transport pathway, to provide an effective solution to the delivery and toxicity problem of systemically administered chemotoxins. Work will be based on the current proprietary mouse monoclonal antibody that targets a truncated form of Annexin A1 (mAnnA1) that is concentrated in the endothelial cell caveolae of tumor vasculature. Previous work has demonstrated mAnnA1 to be the first antibody to penetrate solid tumors actively, rapidly, and specifically, and concentrate attached cargo directly into target tissue. Humanization of mAnnA1 is expected to yield a variant with similar binding affinity, stability, and purity that can then be used for further commercial development to generate novel immunoconjugates to treat many forms of primary and metastatic cancers. We hypothesize that enhancing precision delivery will confer significant survival advantages for patients through the antibody's ability to concentrate chemotoxins directly into solid tumors and metastatic lesions, thus sparing exposure to healthy tissues. The overall objective of this Phase I application is to advance translational studies of mAnnA1 through development and selection of a humanized antibody with optimized binding affinity and therapeutic potential. In Aim 1, antibody humanization will proceed using CDR grafting technology, producing 9-16 variants. Selection of hAnnA1 candidates with binding affinities similar to mAnnA1 will be performed using ELISA and surface plasmon resonance analysis. In Aim 2, hAnnA1 variants will be further screened by their ability to retain key biophysical properties (binding affinity, stability, purity) after chemical conjugation. The therapeutic potencies of the top 2 variants, in the form of therapeutically active immunoconjugates, will be validated in vivo using a metastatic model of breast cancer. The proposed study is highly significant because a first-in-class humanized AnnA1 antibody with minimized immunogenicity could deliver therapeutic cargo precisely across in vivo barriers and concentrate them inside tumors, and thus obviate the current reliance on passive transvascular delivery. If successful, it will create the first precision drug delivery platform based on the caveolae pumping system. The resulting lead hAnnA1 antibody will be advanced in an STTR Phase II project for further commercial and preclinical development, pharmacological and toxicological testing, and other studies needed for IND filing and clinical utility. Potential partnering or out-licensing points for pharmaceutical company facilitation of commercialization of hAnnA1 can occur after each completed phase of the overall project.